The mycobacteria are a genus of bacteria that are characterized as acid-fast, non-motile, gram-positive bacillus. The genus comprises many species including Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. leprae, M. microti, M. scrofulaceum, M. paratuberculosis, and M. tuberculosis. Some of the mycobacteria are pathogenic to both humans and animals, in particular M. tuberculosis, M. leprae, and M. bovis. Other mycobacterial species are not normally pathogenic, but cause opportunistic infections in immunocompromised individuals, such as AIDs patients. For example, infection by M. kansasii, M. avium, and M. intracellulare can cause severe lung disease in subjects in whom the immune system is suppressed or compromised. In fact, for the first time since 1953, reported cases of mycobacterial infections are increasing in the United States; many of these cases are related to the AIDS epidemic.
Conventional laboratory diagnosis of mycobacteria is based on acid-fast staining and cultivation of the organism, followed by biochemical assays. As a result of the slow growth and long generation time of mycobacteria, accurate laboratory diagnosis of mycobacteria by conventional techniques can take as long as six weeks. Automated culturing systems such as the BACTEC.TM. system (Becton Dickinson Microbiology Systems, Sparks, Md.) can decrease the time for identification of mycobacteria to one to two weeks. Nevertheless, there still exists a need in the art to reduce the time required for accurate diagnosis of mycobacteria to less than a week, preferably to about one day.
Nucleic acid based diagnostic assays, such as Southern hybridization, offer rapid results, usually in less than one day. PCR-based methods for identifying mycobacteria are even more sensitive and can often provide results within hours. However, nucleic acid based methodologies for diagnosing mycobacteria are often fraught with drawbacks. Most of these methods are costly, are available for only a few species of mycobacteria, and can resolve only one species per sample tested. Moreover, nucleic acid based assays require the development of oligonucleotide probes or primers that are specific for the genus Mycobacterium or for a particular species of mycobacteria.
Conventional laboratory identification of the mycobacterial species M. kansasii is based upon growth characteristics and biochemical testing. The biochemical profile of M. kansasii includes catalase production, urease activity, TWEEN.TM. hydrolysis, nitrate reduction, and photochromogenicity (i.e., the bacterium produces pigment when exposed to light). Several other species of mycobacterium show similar biochemical properties to M. kansasii, and photochromogenicity is usually relied upon for conclusive identification of M. kansasii. Determination of photochromogenicity is often problematic because it requires a pure organism culture, and this trait is variable, subjective and difficult to determine reliably.
To obviate the problems attendant to conventional diagnosis of M. kansasii, there have been attempts to develop nucleic acid based diagnostic methods using species-specific hybridization or nucleic acid amplification with M. kansasii-specific oligonucleotide primers.
Z. H. Huang et al. (J. Clin. Microbiol. 29, 2125 (1991)) disclose a DNA probe (pMK1-9) from a M. kansasii genomic library. The pMK1-9 probe hybridizes to M. kansasii DNA, but it also cross-hybridizes with other species of mycobacteria. In addition, this probe fails to detect one genetically distinct sub-group of M. kansasii. Huang et al. did not report the nucleotide sequence of pMK1-9, nor was the gene from which it was derived identified. B. C. Ross et al. (J. Clin. Microbiol. 30, 2930 (1992)) concerns the identification of M. kansasii using the pMK1-9 probe and a commercial DNA probe that specifically hybridized to the M. kansasii rRNA gene (ACCU-PROBE.TM., Gen-Probe, San Diego, Calif.). Ross et al. reported that both the pMK1-9 probe and the ACCU-PROBE.TM. failed to detect a significant number of M. kansasii strains. Tortoli et al. (Eur. J. Clin. Microbiol. Infect. Dis. 13, 264 (1994)) also evaluated the efficacy of using the ACCU-PROBE.TM. to detect M. kansasii. These investigators found the ACCU-PROBE.TM. was 100% species-specific, showing no cross-reactivity with other mycobacterial species, but it only detected 73% of the M. kansasii strains tested, possibly as a result of the genetic heterogeneity among the strains.
M. Yang et al. (J. Clin. Microbiol. 31, 2769 (1993)) derived an M. kansasii specific DNA hybridization probe (p6123) from a clinical isolate of M. kansasii. The p6123 probe hybridized to all M. kansasii strains tested, including the sub-group that Ross et al. (supra) found to be pMK1-9 negative. U.S. Pat. No. 5,500,341 to Spears discloses M. kansasii-specific amplification primers derived from the p6123 probe.
B. Boddinghaus et al. (J. Clin. Microbiol. 28, 1751 (1990)) disclose Mycobacterium genus-specific oligonucleotides derived from 16S rRNA sequences that specifically amplify and hybridize to mycobacterial DNA.
T. Rogall et al. (J. Gen. Microbiol. 136, 1915 (1990)) used PCR amplification of a region of the 16S rRNA gene followed by direct sequencing to identify various mycobacterial species. However, this method could not distinguish M. kansasii from M. gastri because the sequences of the 16S rRNA gene in these two species is identical, despite their differing phenotypic characteristics.
Hughes et al. (J. Clin. Microbiol. 31, 3216 (1993)) used PCR to amplify the 16S rRNA gene followed by either restriction enzyme analysis or direct cycle sequencing to identify various mycobacterial species. Hughes et al. also found that these methods could not differentiate between M. kansasii and M. gastri. Kirschner et al. (J. Clin. Microbiol. 31, 2882 (1993)) reported similar results. Kirschner et al. also disclose that M. kansasii and M. gastri can be distinguished by supplementing the nucleic acid based diagnostic methods with a photochromogenecity test. Id. at 2885.
M. Vaneechoutte et al., (J. Clin. Microbiol 31, 2061 (1993)) teaches a method of identifying specific mycobacterial species, including M. kansasii, by PCR amplification of the 16S rDNA combined with restriction analysis of the amplification products. This technique allows the positive identification of M. kansasii within one day. Vaneechoutte et al. did not evaluate whether this technique could identify M. gastri or whether it could distinguish M. kansasii from M. gastri.
Accordingly, there remains a need in the art for rapid, accurate and sensitive methods of identifying M. kansasii.